The present invention provides aminoalkoxy carbazole derivatives, and more specifically, provides compounds of formula (I) described herein below. These compounds are 5-HT ligands, and are useful for treating diseases wherein modulation of 5-HT activity is desired.
Many diseases of the central nervous system are influenced by the adrenergic, the dopaminergic, and the serotonergic neurotransmitter systems. For example, serotonin has been implicated in a number of diseases and conditions which originate in the central nervous system. These include diseases and conditions related to sleeping, eating, perceiving pain, controlling body temperature, controlling blood pressure, depression, anxiety, schizophrenia, and other bodily states. R. W. Fuller, Biology of Serotonergic Transmission, 221 (1982); D. J. Boullin, Serotonin in Mental Abnormalities 1:316 (1978); J. Barchas, et al., Serotonin and Behavior, (1973). Serotonin also plays an important role in peripheral systems, such as the gastrointestinal system, where it has been found to mediate a variety of contractile, secretory, and electrophysiologic effects.
As a result of the broad distribution of serotonin within the body, there is a tremendous interest in drugs that affect serotonergic systems. In particular, receptor-specific agonists and antagonists are of interest for the treatment of a wide range of disorders, including anxiety, depression, hypertension, migraine, obesity, compulsive disorders, schizophrenia, autism, neurodegenerative disorders (e.g. Alzheimer""s disease, Parkinsonism, and Huntington""s chorea), and chemotherapy-induced vomiting. M. D. Gershon, et al., The Peripheral Actions of 5-Hydroxytryptamine, 246 (1989); P. R. Saxena, et al., Journal of Cardiovascular Pharmacology, 15:Supplement 7 (1990).
The major classes of serotonin receptors (5-HT1-7) contain fourteen to eighteen separate receptors that have been formally classified. See Glennon, et al., Neuroscience and Behavioral Reviews, 1990, 14, 35; and D. Hoyer, et al. Pharmacol. Rev. 1994, 46, 157-203. Recently discovered information regarding subtype identity, distribution, structure, and function suggests that it is possible to identify novel, subtype specific agents having improved therapeutic profiles (e.g. fewer side effects).
For example, the 5-HT6 receptor was identified in 1993 (Monsma et al. Mol. Pharmacol. 1993, 43, 320-327 and Ruat, M. et al. Biochem. Biophys. Res. Com. 1993, 193, 269-276). Several antidepressants and atypical antipsychotics bind to the 5-HT6 receptor with high affinity and this binding may be a factor in their profile of activities (Roth et al. J. Pharm. Exp. Therapeut. 1994, 268, 1403-1410; Sleight et al. Exp. Opin. Ther. Patents 1998, 8, 1217-1224; Bourson et al. Brit. J. Pharm. 1998, 125, 1562-1566; Boess et al. Mol. Pharmacol. 1998, 54, 577-583; Sleight et al. Brit. J. Pharmacol. 1998, 124, 556-562). In addition, the 5-HT6 receptor has been linked to generalized stress and anxiety states (Yoshioka et al. Life Sciences 1998, 17/18, 1473-1477). Together these studies and observations suggest that compounds that antagonize the 5-HT receptor will be useful in treating disorders of the central nervous system.
Compounds of the present invention are 5-HT ligands (e.g. receptor-specific agonists or antagonists). Thus they are useful for treating diseases wherein modulation of 5-HT activity is desired. Specifically, the compounds of this invention are useful in the treatment of psychosis, paraphrenia, psychotic depression, mania, schizophrenia, schizophreniform disorders, schizoaffective disorder, delusional disorder, panic disorder, a phobia, obsessive compulsive disorder, post-traumatic-stress syndrome, immune system depression, a stress induced problem with the urinary, a stress related disease such as anxiety, migraine headache, drug addiction, convulsive disorders, personality disorders, post-traumatic stress syndrome, alcoholism, panic attacks, obsessive-compulsive disorders, sleep disorders, disorders of the gastrointestinal or cardiovascular system (e.g., stress incontinence), neurodegenerative disorders, autism, chemotherapy-induced vomiting, hypertension, cluster headaches, sexual dysfunction in a mammal (e.g. a human), addictive disorder and withdrawal syndrome, an adjustment disorder, an age-associated learning and mental disorder, anorexia nervosa, apathy, an attention-deficit disorder due to general medical conditions, attention-deficit hyperactivity disorder, behavioral disturbance (including agitation in conditions associated with diminished cognition (e.g., dementia, mental retardation or delirium)), bipolar disorder, bulimia nervosa, chronic fatigue syndrome, conduct disorder, cyclothymic disorder, dysthymic disorder, fibromyalgia and other somatoform disorders, an inhalation disorder, an intoxication disorder, movement disorder (e.g., Huntington""s disease or Tardive Dyskinesia), oppositional defiant disorder, peripheral neuropathy, post-traumatic stress disorder, premenstrual dysphoric disorder, a psychotic disorder (brief and long duration disorders, psychotic disorder due to medical condition, psychotic disorder NOS), mood disorder (major depressive or bipolar disorder with psychotic features) seasonal affective disorder, a specific developmental disorder, agitation disorder, selective serotonin reuptake inhibition (SSRI) xe2x80x9cpoop outxe2x80x9d syndrome or a Tic disorder (e.g., Tourette""s syndrome). More specifically, the compounds of this invention are useful to treat psychotic, affective, vegetative, and psychomotor symptoms of schizophrenia or the extrapyramidal motor side effects of an antipsychotic drug. This last action will allow higher doses of antipsychotics to be used and thus greater antipsychotic efficacy to be obtained as a result of a reduction in side effects. The compounds of this invention are also useful in the modulation of eating behavior and thus are useful in treating excess weight and associated morbidity and mortality.
International Publication No. WO95/03296 discloses carbazole and dibenzofuran hapten compounds.
Abstract of International Publication No. WO/9747601 discloses new heterocyclic compounds useful for treating schizophrenia.
European Patent Application EP 839806 discloses tricyclic compounds useful for inhibiting sPLA2 mediated release of fatty acids for conditions such as septic shock.
U.S. Pat. No. 5,668,167 discloses carbazole derivatives useful for treating microbial infections.
U.S. Pat. No. 4,503,067 discloses carbazolyl-(4)-oxypropanolamine compounds useful for treating cardiac diseases.
U.S. Pat. No. 3,932,424 discloses carbazoles useful as antiviral agents.
U.S. Pat. No. 3,896,145 discloses carbazoles useful as antiinflammatory, analgesic, and anti-rheumatic agents.
U.S. Pat. No. 3,759,948 discloses tricyclic carboxylic acid and ester derivatives useful as anti-inflammatory, anti-pyretic and analgesic agents.
DE 4330175 discloses beta carbolines useful for treating Parkinson""s disease, Alzheimer""s disease, senile dementia, epilepsy, schizophrenia, migraine etc.
Abstract of Japanese Patent No. 06228095 discloses carbazole derivatives useful for treating ischaemic encephalopathy.
Abstract of French Patent No. 2516512 discloses pyrido-indole derivatives useful for treating cardiac rhythm diseases.
The present invention provides a compound of formula I 
wherein
R1 is
(a) H,
(b) halo, or
(c) C1-6 alkyl;
R2 is
(a) H,
(b) halo,
(c) xe2x80x94OH,
(d) xe2x80x94CN,
(e) xe2x80x94CF3,
(f) xe2x80x94O(C1-6)alkyl,
(g) C1-6 alkyl,
(h) C3-6 cycloalkyl,
(i) xe2x80x94NR5R6,
(j) xe2x80x94CONR5R6,
(k) xe2x80x94SO2NR5R6,
(l) xe2x80x94COOR7,
(m) xe2x80x94OCF3, or
(n) phenyl, optionally substituted with halo, OH, O(C1-4) alkyl, or C1-6 alkyl; each R3 is independently
(a) H,
(b) C1-6 alkyl, or
(c) C3-6 cycloalkyl;
R4 is
(a) aryl, or
(b) heteroaryl;
R5 and R6 are independently
(a) H,
(b) C1-6 alkyl, or
(c) C3-6 cycloalkyl;
R7 is
(a) H,
(b) C1-6 alkyl, or
(c) (C1-3 alkyl)-phenyl wherein phenyl may be substituted with R3;
R8 and R9 are independently
(a) H,
(b) C1-6 alkyl, optionally substituted with aryl, hetroaryl, or C3-6 cycloalkyl,
(c) C2-6 alkenyl,
(d) C3-6 cycloalkyl,
(e) C2-6 alkyl substituted with R10,
(f) xe2x80x94CHO, provided that only one of the R8 and R9 is CHO, the other one is H,
(g) aryl,
(h) heterocyclic, which is bonded via carbon atom to the nitrogen to which it is attached, or
(i) R8 and R9 taken together with the nitrogen to which they are attached form a heterocyclic ring wherein the heterocyclic ring may have one to two additional heteroatoms selected from the group consisting of oxygen, sulfur and N(Y) and wherein the carbon atoms of the heterocyclic ring is optionally substituted with one or two R14;
R10 is
(a) xe2x80x94OH,
(b) xe2x80x94O(C1-4 alkyl), optionally alkyl is substituted with OH,
(c) xe2x80x94O(C1-4 alkyl)-NR11R12,
(d) heterocyclic, or
(e) xe2x80x94CO2R5,
R11 and R12 are independently,
(a) H, or
(b) C1-4 alkyl;
aryl is phenyl or naphthyl, optionally substituted with one or more R13; heteroaryl is a radical of a five- or six-membered monocyclic aromatic ring having one or two heteroatoms each selected from the group consisting of oxygen, sulfur, and N(X), or a radical of a nine- or ten-membered ortho-fused bicyclic aromatic ring having one, two or three heteroatoms each selected from the group consisting of oxygen, sulfur, and N(X); wherein carbon atoms of heteroaryl may be substituted with R13;
heterocyclic is a radical of a five-, six-, or seven-membered partially-saturated or unsaturated heterocyclic ring having one, two or three heteroatoms selected from the group consisting of oxygen, sulfur and N(Y) wherein the carbon atoms of the heterocyclic ring may be substituted with R14;
X is absent, H or C1-4 alkyl;
Y is
(a) H,
(b) C1-6 alkyl, optionally substituted with aryl or hetroaryl,
(c) C3-6 cycloalkyl, or
(d) C2-6 alkyl substituted with xe2x80x94OH, xe2x80x94O(C1-4 alkyl), xe2x80x94O(C1-4 alkyl)-NR11R12, xe2x80x94CO2R5, or NHCHO, or
(e) xe2x80x94OH;
R13 is
(a) halo,
(b) xe2x80x94OH,
(c) xe2x80x94CN,
(d) xe2x80x94CF3,
(e) xe2x80x94O(C1-6)alkyl,
(f) C1-6 alkyl,
(g) C3-6 cycloalkyl,
(h) xe2x80x94NR5R6,
(i) xe2x80x94CONR5R6,
(j) xe2x80x94SO2NR5R6,
(k) xe2x80x94COOR7,
(l) xe2x80x94OCF3, or
(m) phenyl, optionally substituted with halo, OH, O(C1-4) alkyl, or C1-6 alkyl;
R14 is
(a) C1-6 alkyl,
(b) C3-6 cycloalkyl,
(c) C2-6 alkyl substituted with xe2x80x94OH, xe2x80x94O(C1-4 alkyl), xe2x80x94O(C1-4 alkyl)-NR11R12, or xe2x80x94CO2R5,
(d) xe2x80x94OH, or
(e) oxo (xe2x95x90O);
m is 1, 2, 3 or 4;
n is 1, 2, 3, or 4;
C3-6 cycloalkyl in each of the above definitions, may be each and independently substituted with xe2x80x94OH, C1-4 alkyl, or oxo (xe2x95x90O), and with the following provisos:
(a) when R4 is 4-fluorophenyl, n is 1, m is 1, each R3 is independently hydrogen, R8 and R9 is independently xe2x80x94CH2CH3, then R2 cannot be fluoro or chloro at the C-6 position of formula I;
(b) when n is 1, m is 1, R2, R3, R8 or R9 is hydrogen, R4 is 4-thiazolyl, then said 4-thiazolyl cannot be substituted with 4-chlorophenyl;
(c) when n is 1, m is 1, R2, R3, R8 or R9 is hydrogen, then R4 is not 4-pyridyl;
(d) when n is 1, m is 1, R2, R3, R8 or R9 is hydrogen, then R4 is not 2-bromophenyl or 4-bromophenyl.
In another aspect, the present invention also provides:
a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient (the composition preferably comprises a therapeutically effective amount of the compound or salt),
a method for treating a disease or condition in a mammal (e.g. a human) wherein a 5-HT receptor is implicated and modulation of a 5-HT function is desired comprising administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof to the mammal,
a method for treating or preventing anxiety, obesity, depression, schizophrenia, a stress related disease (e.g. general anxiety disorder), panic disorder, a phobia, obsessive compulsive disorder, post-traumatic-stress syndrome, immune system depression, a stress induced problem with the gastrointestinal or cardiovascular system, eating disorders or sexual dysfunction in a mammal (e.g. a human) comprising administering a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof to the mammal,
a method of treating or preventing diseases or disorders of the central nervous system such as: psychosis, paraphrenia, psychotic depression, mania, schizophrenia, schizophreniform disorders, schizoaffective disorder, delusional disorder, panic disorder, a phobia, obsessive compulsive disorder, post-traumatic-stress syndrome, immune system depression, a stress induced problem with the urinary, a stress related disease such as anxiety, migraine headache, drug addiction, convulsive disorders, personality disorders, post-traumatic stress syndrome, alcoholism, panic attacks, obsessive-compulsive disorders, sleep disorders, disorders of the gastrointestinal or cardiovascular system (e.g., stress incontinence), neurodegenerative disorders, autism, chemotherapy-induced vomiting, hypertension, cluster headaches, sexual dysfunction in a mammal (e.g. a human), addictive disorder and withdrawal syndrome, an adjustment disorder, an age-associated learning and mental disorder, anorexia nervosa, apathy, an attention-deficit disorder due to general medical conditions, attention-deficit hyperactivity disorder, behavioral disturbance (including agitation in conditions associated with diminished cognition (e.g., dementia, mental retardation or delirium)), bipolar disorder, bulimia nervosa, chronic fatigue syndrome, conduct disorder, cyclothymic disorder, dysthymic disorder, fibromyalgia and other somatoform disorders, an inhalation disorder, an intoxication disorder, movement disorder (e.g., Huntington""s disease or Tardive Dyskinesia), oppositional defiant disorder, peripheral neuropathy, post-traumatic stress disorder, premenstrual dysphoric disorder, a psychotic disorder (brief and long duration disorders, psychotic disorder due to medical condition, psychotic features), seasonal affective disorder, a specific developmental disorder, agitation disorder, selective serotonin reuptake inhibition (SSRI) xe2x80x9cpoop outxe2x80x9d syndrome or a Tic disorder (e.g., Tourette""s syndrome), comprising administering to a mammal (e.g. a human) in need of such treatment, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof,
a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in medical diagnosis or therapy (e.g. the treatment or prevention of 5-HT related central nervous system diseases or disorders),
the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating or preventing 5-HT related central nervous system diseases or disorders such as anxiety, obesity, depression, schizophrenia, a stress related disease (e.g. general anxiety disorder), panic disorder, a phobia, obsessive compulsive disorder, post-traumatic-stress syndrome, immune system depression, a stress induced problem with the gastrointestinal or cardiovascular system, or sexual dysfunction in a mammal (e.g. a human), and
a method for modulating 5-HT (e.g. 5-HT6) receptor function, comprising contacting (in vitro or in vivo) the receptor with an effective inhibitory amount of a compound of formula I, or a pharmaceutically acceptable salt thereof.
The invention also provides novel intermediates and processes disclosed herein that are useful for preparing compounds of formula I.
The compounds of the present invention are generally named according to the IUPAC or CAS nomenclature system. Abbreviations which are well known to one of ordinary skill in the art may be used (e.g. xe2x80x9cPhxe2x80x9d for phenyl, xe2x80x9cMexe2x80x9d for methyl, xe2x80x9cEtxe2x80x9d for ethyl, xe2x80x9chxe2x80x9d for hour or hours and xe2x80x9crtxe2x80x9d for room temperature).
The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Ci-j indicates a moiety of the integer xe2x80x9cixe2x80x9d to the integer xe2x80x9cjxe2x80x9d carbon atoms, inclusive. Thus, for example, C1-7alkyl refers to alkyl of one to seven carbon atoms, inclusive.
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
The following definitions are used, unless otherwise described.
Halo is fluoro, chloro, bromo, or iodo.
Alkyl denotes both straight and branched groups; but reference to an individual radical such as xe2x80x9cpropylxe2x80x9d embraces only the straight chain radical, a branched chain isomer such as xe2x80x9cisopropylxe2x80x9d being specifically referred to. Specifically, C1-7 alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl.
Alkenyl denotes both straight and branched groups have at least one double bond.
C3-6 cycloalkyl denotes a cycloalkyl having three to six carbon atoms. Specifically, C3-6 cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.
Aryl denotes a phenyl or a naphthyl radical. Optionally, aryl is substituted with one or more halo, OH, CN, CF3, O(C1-6)alkyl, C1-6 alkyl, C3-6 cycloalkyl, NR5R6, CONR5R6, SO2NR5R6, COOR7, OCF3, or phenyl which in turn may be substituted with halo, OH, O(C1-4) alkyl, or C1-6 alkyl. R5 and R6 are the same as summarized above.
Heteroaryl denotes a radical of a five- or six-membered monocyclic aromatic ring having one or two heteroatoms each selected from the group consisting of oxygen, sulfur, and N(X), or a radical of a nine- or ten-membered ortho-fused bicyclic aromatic ring having one, two or three heteroatoms each selected from the group consisting of oxygen, sulfur, and N(X); wherein X is absent, H or C1-4 alkyl; wherein carbon atoms of heteroaryl may be substituted with one or more halo, OH, CN, CF3, O(C1-6)alkyl, C1-6 alkyl, C3-6 cycloalkyl, NR5R6, CONR5R6, SO2NR5R6, COOR7, OCF3, or phenyl which in turn may be substituted with halo, OH, O(C1-4) alkyl, or C1-6 alkyl. R5, R6 and R7 are the same as summarized above. Examples of heteroaryl are pyridyl, thiophene, benzothiophene, benzofuran, benzimidazole, imidazole or thiazole.
Heterocyclic is a radical of a five-, six-, or seven-membered partially-saturated or unsaturated heterocyclic ring having one, two or three heteroatoms selected from the group consisting of oxygen, sulfur and N(Y) wherein the carbon atoms of the heterocyclic ring are optionally substituted with R14. Y and R14 are the same as summarized above. Examples of heterocyclic is azetidyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1-benzyl-piperidinyl, 1-methyl-piperidinyl, dioxolane, imidazolidine, oxazolidinyl, oxathiolane, 4-hydroxyl-1-piperidinyl, 4-ethanol-1-piperazinyl-, 4-ethylformamide-1-piperazinyl-, or 4-methyl-1-piperazinyl.
Pharmaceutically acceptable salts denotes acid addition salts useful for administering the compounds of this invention and include hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, mesylate, maleate, malate, succinate, tartrate, citric acid, 2-hydroxyethyl sulfonate, fumarate, methanesulfonic acid salt and etc. Specifically, pharmaceutically acceptable salts can be maleate, methanesulfonic acid salt and etc.
Mammal denotes human and animals.
A specific value for R1 is H, halo, or C1-6 alkyl.
A specific value for R1 is H.
A specific value for R2 is H, halo, xe2x80x94OH, xe2x80x94CN, xe2x80x94CF3, xe2x80x94O(C1-6)alkyl, C1-6 alkyl, C3-6 cycloalkyl, xe2x80x94NR5R6, xe2x80x94CONR5R6, xe2x80x94SO2NR5R6, xe2x80x94COOR7, or phenyl which may be substituted with halo, xe2x80x94OH, xe2x80x94O(C1-4) alkyl, or C1-6 alkyl; wherein R5 and R6 is H, C1-6 alkyl, or C3-6 cycloalkyl; wherein R7 is C1-6 alkyl, or (C1-3 alkyl)-phenyl wherein phenyl may be substituted with R2.
A specific value for R2 is H, halo, or C1-6 alkyl.
A specific value for R2 is H, chloro, fluoro, or methyl.
A specific value for R2 is H.
A specific value for R2 is fluoro or methyl.
A specific value for each R3 is independently H, C1-6 alkyl, or C3-6 cycloalkyl.
A specific value for each R3 is independently H.
A specific value is wherein R8 and R9 are independently H, C1-6 alkyl (optionally substituted with aryl, heteroaryl or C3-6 cycloalkyl), C2-6 alkenyl, C3-6 cycloalkyl, C2-6 alkyl substitute with R10, xe2x80x94CHO (provided that only one of the R8 and R9 is xe2x80x94CHO, the other one is hydrogen), aryl, heterocyclic wherein heterocyclic is bonded via carbon atom to the nitrogen to which it is attached, or R8 and R9 taken together with the nitrogen to which they are attached form a heterocyclic ring wherein the heterocyclic ring may have one to two additional heteroatoms selected from the group consisting of oxygen, sulfur and N(Y) and wherein the carbon atoms of the heterocyclic ring are optionally substituted with one or two R13; wherein R10, and Y are as defined above, wherein each C3-6 cycloalkyl is optionally substituted with xe2x80x94OH, C1-4 alkyl, or oxo.
A specific value is wherein R8 is H, and R9 is H, C1-6 alkyl, C2-4 alkenyl, C1-6 alkyl substituted with phenyl, wherein phenyl is optionally substituted with fluoro or chloro.
A specific value is wherein R8 is H, and R9 is C1-6 alkyl substituted with C3-6 cycloalkyl, wherein cycloalkyl is optionally substituted with xe2x80x94H, C1-4 alkyl or oxo.
A specific value is wherein R8 is H, and R9 is C2-6 alkyl substituted with xe2x80x94OH, xe2x80x94O(C1-4 alkyl), xe2x80x94O(C1-4 alkyl-OH) or xe2x80x94CO2C1-4 alkyl.
A specific value is wherein R8 is H, and R9 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, all of which may be substituted with xe2x80x94OH, C1-4 alkyl, oxo, or xe2x80x94CHO.
A specific value is wherein R8 is H, and R9 is C1-6 alkyl substituted with phenyl, pyridyl, thiophene, benzothiophene, benzofuran, benzimidazole, imidazole or thiazole.
A specific value is wherein R8 is H, and R9 is heterocyclic wherein heterocyclic is bonded via carbon atom to the nitrogen to which it is attached.
A specific value is wherein R8 is H, and R9 is pyridyl methyl, benzimidazole methyl, or 1-benzyl-piperidinyl.
A specific value is wherein R8 is H; and R9 is C2-6 alkyl substituted with azetidyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1-benzyl-piperidinyl, 1-methyl-piperidinyl, dioxolane, imidazolidine, oxazolidinyl, oxathiolane, 4-hydroxyl-1-piperidinyl, 4-ethanol-1-piperazinyl-, 4-ethylformamide-1-piperazinyl-, or 4-methyl-1-piperazinyl.
A specific value is wherein R8 and R9 taken together with the nitrogen to which they are attached form a heterocyclic ring wherein the heterocyclic ring may have one to two additional heteroatoms selected from the group consisting of oxygen, sulfur and N(Y), wherein Y is the same as defined in claim 1.
A specific value is wherein R8 and R9 taken together with the nitrogen to which they are attached form 4-morpholinyl, 1-pyrrolidinyl, 1-piperidinyl, 1-piperazinyl, 4-hydroxyl-1-piperidinyl, 4-ethanol-1-piperazinyl-, 4-ethylformamide-1-piperazinyl-, or 4-methyl-1-piperazinyl.
A specific value for R8 and R9 are independently H, methyl, ethyl, propyl, 1-propanol, 2-propenyl, 1-pentanol, 2-methyl-1-propanol, 2-butanol, 1-ethanol, ethoxyl-1-ethanol, xe2x80x94CH2CH2CO2ethyl, 2-methoxyethyl, 4-chlorophenethyl, or 4-fluorophenethyl.
A specific value is wherein R8 and R9 are both hydrogen atoms.
A specific value is wherein R8 is H; and R9 is methyl.
A specific value is wherein R8 and R9 taken together with the nitrogen to which they are attached form 4-methyl-1-piperazinyl.
A specific value for R4 is aryl, or heteroaryl; wherein aryl or heteroaryl are as defined as herein above.
A specific value for R4 is phenyl.
A specific value for R4 is pyridyl, thiophene, benzothiophene, benzofuran, benzimidazole, imidazole, thiazole pyridyl, thiophene, benzothiophene, benzofuran, benzimidazole, imidazole or thiazole.
A specific value for R4 is 2-methyl-1,3-thiazol-4-yl, or 5-chloro-1-benzothiophn-3-yl.
A specific value for m is one.
Examples of the present invention includes:
a) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
b) N-{2-[(9-benzyl-8-chloro-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
c) N-(2-{[8-chloro-9-(4-fluorobenzyl)-9H-carbazol-4-yl]oxy}ethyl)-N,N-diethylamine,
d) N-{2-[(9-benzyl-8-methyl-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
e) N,N-diethyl-N-(2-{[9-(4-fluorobenzyl)-8-methyl-9H-carbazol-4-yl]oxy}ethyl)amine,
f) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N-(2-pyridinylmethyl)amine,
g) N-{2-[(9-benzyl-8-fluoro-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
h) 9-benzyl-4-[2-(4-morpholinyl)ethoxy]-9H-carbazole,
i) 2-(4-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-1-piperazinyl)-1-ethanol,
j) 3-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)-1-propanol,
k) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-2-propen-1-amine
l) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-3-(4-morpholinyl)-1-propanamine,
m) 5-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)-1-pentanol,
n) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-1-propanamine,
o) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N-propyl-1-propanamine,
p) 1-benzyl-N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-4-piperidinamine,
q) 2-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)-2-methyl-1-propanol,
r) 2-[(9-benzyl-9H-carbazol-4-yl)oxy]-N-(4-chlorophenethyl)-1-ethanamine,
s) 2-[(9-benzyl-9H-carbazol-4-yl)oxy]-N-(cyclohexylmethyl)-1-ethanamine,
t) 2-[(9-benzyl-9H-carbazol-4-yl)oxy]-N-[2-(4-morpholinyl)ethyl]-1-ethanamine,
u) 1-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)-2-butanol,
v) 2-[(9-benzyl-9H-carbazol-4-yl)oxy]-N-(4-fluorophenethyl)-1-ethanamine,
w) 2-[2-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)ethoxy]-1-ethanol,
x) (1S,2S)-2-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)cyclohexanol,
y) ethyl 3-({2-[(9-benzyl-9H-carbazol4-yl)oxy]ethyl}amino)propanoate,
z) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}cyclobutanamine,
aa) 2-(4-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-1-piperazinyl)ethylformamide,
bb) N-(1H-benzimidazol-2-ylmethyl)-2-[(9-benzyl-9H-carbazol-4-yl)oxy]-1-ethanamine,
cc) 1-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-4-piperidinol,
dd) 9-benzyl-4-[2-(4-methyl-1-piperazinyl)ethoxy]-9H-carbazole,
ee) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N-cyclopropylamine,
ff) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N,N-dimethylamine,
gg) N-{2-[(9-benzyl-9H-carbazol -4-yl)oxy]ethyl}formamide,
hh) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N-methylamine, or its maleic acid salt,
ii) 2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethylamine,
jj) N-{2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}-N-(2-methoxyethyl)aamine,
kk) N-ethyl-N-{[(1-phenyl-1,2-dihydro[1,4]oxazino[2,3,4-jk]carbazol-7-yl)oxy]ethyl}amine, or its maleic acid salt,
ll) 9-benzyl-4-[2-(1-pyrrolidinyl)ethoxy]-9H-carbazole,
mm) 9-benzyl-4-[2-(1-piperidinyl)ethoxy]-9H-carbazole,
nn) 9-benzyl-4-[2-(1-piperazinyl)ethoxy]-9H-carbazole,
oo) 2-[(9-benzyl-8-fluoro-9H-carbazol-4-yl)oxy]ethylamine,
pp) N,N-diethyl-N-(2-{[8-fluoro-9-(4-fluorobenzyl)-9H-carbazol-4-yl]oxy}ethyl)amine,
qq) N-{2-[(9-benzyl-6-chloro-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
rr) N-{2-[(9-benzyl-6-fluoro-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
ss) N-{2-[(9-benzyl-6-methyl-9H-carbazol-4-yl)oxy]ethyl}-N,N-diethylamine,
tt) 2-[(9-benzyl-6-methyl-9H-carbazol-4-yl)oxy]ethylamine,
uu) N-{2-[(9-benzyl-6-methyl-9H-carbazol-4-yl)oxy]ethyl}-N-methylamine,
vv) N,N-diethyl-N-(2-{[9-(4-fluorobenzyl)-6-methyl-9H-carbazol-4-yl]oxy}ethyl)amine,
ww) 2-({2-[(9-benzyl-9H-carbazol-4-yl)oxy]ethyl}amino)-1-ethanol or its maleic acid salt,
xx) 2-({9-[(5-Chloro-1-benzothiophen-3-yl)methyl]-9H-carbazol-4-yl}oxy)ethylamime or its methane sulfonate salt,
yy) 2-({9-[(2-Methyl-1,3-thiazol-4-yl)methyl]-9H-carbazol-4-yl}oxy)ethylamine or its methane sulfonate salt,
zz) 2-[(9-benzyl-3-chloro-9H-carbazol-4-yl)oxy]ethylamine, methanesulfonate salt,
aaa) 2-{[9-(3-bromobenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
bbb) 2-{[9-(3-fluorobenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
ccc) 2-{[9-(4-methylbenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
ddd) 2-{[9-(2-fluorobenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
eee) 2-{[9-(3-methoxybenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
fff) 2-{[9-(3,5-dimethoxybenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
ggg) 2-{[9-(3-methylbenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
hhh) 2-{[9-(2-methylbenzyl)-9H-carbazol-4-yl]oxy}ethylamine, maleic acid salt,
iii) 2-[(9-benzyl-6-methoxy-9H-carbazol-4-yl)oxy]ethylamine, or
jjj) 2-[(9-benzyl-7-methoxy-9H-carbazol-4-yl)oxy]ethylamine.
It will be appreciated by those skilled in the art that compounds of the invention may contain a chiral center, therefore, they may be isolated in optically active or racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically active, polymorphic, tautomeric, or stereoisomeric form, or mixture thereof, of a compound of the invention, which possesses the useful properties described herein. It is well known in the art to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation (using a chiral stationary phase, for example)) and to determine 5-HT6 activity using the standard tests described herein, or using other similar tests which are well known in the art.
The following Schemes describe the preparation of compounds of the present invention. All of the starting materials are commercially available or prepared by procedures described in these schemes or by procedures that would be well known to one of ordinary skill in organic chemistry. The variables used in the Schemes are as defined above or as in the claims.
As shown in Chart A, hydrazines 3 can be prepared from commercially available anilines 1. Aniline 1 is stirred in an acidic medium such as TFA, acetic acid, or aq. sulfuric acid. A nitrite such as sodium nitrite, isoamylnitrite, or n-butylnitrite is added to give nitroso aniline 2. Nitroso aniline 2 is reduced with lithium aluminum hydride in ether or THF to give hydrazine 3. For a discussion of additional methods of preparing 3, see Sandler, S. R.; Karo, W. Organic Functional Group Preparations; Academic Press: New York, 1983; Vol. I, 2nd Ed., pp. 434-465. Hydrazone 4 is prepared from hydrazine 3 and cyclohexane-1,3-dione in solvents such as water, alcohols, or dichloromethane. Hydrazone 4 is then treated under the conditions of the Fischer indole synthesis using an acid and a solvent such as acetic acid, toluene, ethanol, or others, to give tetrahydrocarbazole 5. Alternatively, hydrazine 3 and cyclohexane-1,3-dione may be reacted under Fischer indole conditions to directly give tetrahydrocarbazole 5. Many additional methods for the Fischer indole synthesis are given in Sundberg, R. J.; Indoles, Academic Press: London; 1996, and in Hughes, D. L. Progress in the Fischer Indole Reaction: A Review. Org. Prep. Proceed. Int. 1993, 25, 609-632. The nitrogen of tetrahydrocarbazole 5 is alkylated by methods well known to those versed in the art. For example, treatment of tetrahydrocarbazole 5 with a base such as sodium hydride, followed by an alkyl halide such as benzyl chloride or benzyl bromide, gives benzyl tetrahydrocarbazole 6.
Treatment of compound 6 in a single step using Raney nickel on Pd/carbon in solvents such as cumene, mesitylene, 1,2,3-trimethylbenzene, 1,2,4-trimethylbenzene, decalin, carbitol, or diphenyl ether at temperatures between 130-270xc2x0 C. provides phenol 9 directly. Alternatively, benzyl tetrahydrocarbazole 6 is first treated with a copper (n) halide, preferably CuCl2 or CuBr2, in solvents such as DMF, acetonitrile, EtOAc, chloroform, acetic acid, or acetic acid/water at temperatures between 50 and 120xc2x0 C. to give halo tetrahydrocarbazoles 7 and 8. For a reference to this reaction, see Matsumoto, M.; Ishida, Y.; Watanabe, N. Heterocycles 1985, 23, 165-170. A third useful method is treatment of benzyl tetrahydrocarbazole 6 with pyridinium bromide perbromide or phenyltrimethylammonium tribromide in solvents such as DMF, acetonitrile, or THF to give halo tetrahydrocarbazoles 7 and 8. Halo tetrahydrocarbazoles 7 and 8 may be separated and carried on individually in the next steps, or they may be carried forward as a mixture to phenols 9 and 10 and separated at that time. Halo tetrahydrocarbazoles 7 and 8 (separately or together) are then treated with lithium chloride or lithium bromide (anhydrous LiCl or LiBr is preferred, but hydrated forms also may be used) in the presence of lithium carbonate in a solvent such as DMF at 110-130xc2x0 C. to give phenols 9 and 10.
Charts B and D disclose some of the ways phenols 9 and 10 may be alkylated with various alkylating agents; and Chart C discloses further transformations of carbazole amine 13 to give alkyl amines 15, 16, and 17 directly or after several steps. Which method is used will depend on the type of amine that is desired and on the availability of alkylating agents. For clarity, only 13 is depicted in Chart C, but the reaction scheme applies equally well to 14. In chart D, only phenol 9 is depicted, but the reaction scheme applies equally well to phenol 10.
In Chart B, X refers to a halogen atom. Phenols 9 and 10 are alkylated with chloro or bromoethyl amine in the presence of bases such as sodium hydride, potassium carbonate, cesium carbonate, or sodium carbonate in solvents such as DMF, acetonitrile, or acetone at room temperature to 120xc2x0 C. to give carbazole amines 13 and 14 directly. Alternatively, phenols 9 and 10 are alkylated with chloro- or bromoacetonitrile in the presence of bases such as sodium hydride, potassium carbonate, cesium carbonate, or sodium carbonate in solvents such as DMF, acetonitrile, or acetone at room temperature to 120xc2x0 C. to give nitrites 11 and 12. Reduction of nitrites 11 and 12 with borane in THF or borane-methyl sulfide complex in THF at room temperature to 80xc2x0 C. gives carbazole amines 13 and 14 (R8 and R9 are hydrogen atoms). Other methods for the reduction of the nitrite group to an amine may be found in March, J. Advanced Organic Chemistry, 3rd ed., John Wiley and Sons: New York: 1985.
Chart C discloses further functionalization of carbazole amine 14 by several methods. In Chart C, Q refers to hydrogen, alkyl, or aryl. Z refers to hydrogen or alkyl. One method is acylation with acylating agents such as ethyl formate, acetic anhydride, and the like to give acyl carbazole 15. The carbonyl function of acyl carbazole 15 is reduced to an alkyl group using reagents such as borane in THF or borane-methyl sulfide complex in THF at room temperature to 80xc2x0 C. to give monoalkylamino carbazole 16; or using lithium aluminum hydride in ethereal solvents to effect the reduction to monoalkylamino carbazole 16. A second method is reductive amination of 14 with an equivalent amount of an aldehyde or ketone in the presence of reducing agents such as sodium cyanoborohydride or sodium triacetoxyborohydride in solvents such as dichloromethane, dichloroethane, and THF at 0 to 80xc2x0 C., or Pd/C under a hydrogen atmosphere in solvents such as methanol, ethanol, or ethyl acetate to give monoalkylamino carbazole 16. A third method is alkylation of carbazole amine 14 with alkyl halides or mesylates or tosylates in the presence of base in solvents such as THF, acetonitrile, dichloromethane, DMF and the like using methods well known to those versed in the art to give monoalkylamino carbazole 16.
When dialkylamino carbazole 17 is desired, a second equivalent of the same or a different aldehyde, ketone, or alkylating agent, depending on the method used, is added to monoalkylamino carbazole 16 using the conditons described above. Alternatively, dialkylaminocarbazole 17 may be prepared directly from 14 using two equivalents of the aldehyde, ketone, or alkylating agent.
Chart D describes another method of preparing mono- or dialkylamino carbazole 21, wherein phenol 9 is alkylated by methods well-known to those versed in the art to give carbazole halide 18 or carbazole alcohol 19. In Chart D, L refers to a leaving group such as halo atom or a sulfonate group. The alcohol group of carbazole alcohol 19 is converted to a leaving group with methane sulfonyl halide or toluene sulfonyl halide to give carbazole sulfonate 20. The mesyl or tosyl group of carbazole sulfonate 20 or carbazole halide 18 is then displaced by amines to give amino carbazole 21.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by mixing a compound of the present invention with a suitable acid.
Compounds of the present invention can conveniently be administered in a pharmaceutical composition containing the compound in combination with a suitable excipient. Such pharmaceutical compositions can be prepared by methods and contain excipients which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington""s Pharmaceutical Sciences by E. W. Martin (Mark Publ. Co., 15th Ed., 1975). The compounds and compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal or intramuscular injection), topically, orally, or rectally.
For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The compound is conveniently administered in unit dosage form; for example, containing about 0.05 mg to about 500 mg, conveniently about 0.1 mg to about 250 mg, most conveniently, about 1 mg to about 150 mg of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
The compositions can conveniently be administered orally, sublingually, transdermally, or parenterally at dose levels of about 0.01 to about 150 mg/kg, preferably about 0.1 to about 50 mg/kg, and more preferably about 0.1 to about 30 mg/kg of mammal body weight.
For parenteral administration the compounds are presented in aqueous solution in a concentration of from about 0.1 to about 10%, more preferably about 0.1 to about 7%. The solution may contain other ingredients, such as emulsifiers, antioxidants or buffers.
The exact regimen for administration of the compounds and compositions disclosed herein will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment and, of course, the judgment of the attending practitioner.
Generally, compounds of the invention are 5-HT ligands. The ability of a compound of the invention to bind or act at a 5-HT receptor, or to bind or act selectively at a specific 5-HT receptor subtype can be determined using in vitro and in vivo assays that are known in the art. As used herein, the term xe2x80x9cbind selectivelyxe2x80x9d means a compound binds at least 2 times, preferably at least 10 times, and more preferably at least 50 times more readily to a given 5-HT subtype than to one or more other subtypes. Preferred compounds of the invention bind selectively to one or more 5-HT receptor subtypes.
The ability of a compound of the invention to act as a 5-HT receptor agonist or antagonist can also be determined using in vitro and in vivo assays that are known in the art. The invention provides compounds of formula I that act as either agonists or as antagonists of one or more 5-HT receptor subtypes.
Growth of Cells and Membrane Preparation
Hela cells containing the cloned human 5-HT6 receptor were acquired from Dr. David R. Sibley""s laboratory in National Institute of Health (see Sibley, D. R., J. Neurochemistry, 66, 47-56, 1996). Cells were grown in high glucose Dulbecco""s modified Eagle""s medium, supplemented with L-glutamine, 0.5% sodium pyruvate, 0.3% penicillin-streptomycin, 0.025% G-418 and 5% Gibco fetal bovine serum and then were harvested, when confluent, in cold phosphate-buffered saline.
Harvested intact cells were washed once in cold phosphate-buffered saline. The cells were pelleted and resuspended in 100 ml of cold 50 mM Tris, 5 mM EDTA and 5 mM EGTA, pH 7.4. Homogenization was with a Vir Tishear generator, 4 cycles for 30 seconds each at setting 50. The homogenized cells were centrifuged at 700 RPM (1000xc3x97g) for 10 minutes and the supernatant was removed. The pellet was resuspended in 100 ml of the above buffer and rehomogenized for 2 cycles. The rehomogenized cells were then centrifuged at 700 RPM (1000xc3x97g) for 10 minutes and the supernatant was removed. The combined supernatant (200ml) was centrifuged at 23,000 RPM (80,000xc3x97g) for 1 hour in a Beckman Rotor (42.1 Ti). The membrane pellet was resupended in 50-8-ml of assay buffer containing HEPES 20 mM, MgCl2 10 mM, NaCl 150 mM, EDTA 1 mM, pH 7.4 and stored frozen in aliqouts at xe2x88x9270xc2x0 C.
5-HT6 Receptor Binding Assay
The radioligand binding assay used [3H]-lysergic acid diethylamide (LSD). The assay was carried out in Wallac 96-well sample plates by the addition of 11 xcexcl of the test sample at the appropriate dilution (the assay employed 11 serial concentrations of samples run in duplicate), 11 xcexcl of radioligand, and 178 xcexcl of a washed mixture of WGA-coated SPA beads and membranes in binding buffer. The plates were shaken for about 5 minutes and then incubated at room temperature for 1 hour. The plates were then loaded into counting cassettes and counted in a Wallac MicroBeta Trilux scintillation counter.
Binding Constant (Ki) Determination
Eleven serial dilutions of test compounds were distributed to assay plates using the PE/Cetus Pro/Pette pipetter. These dilutions were, followed by radioligand and the bead-membrane mixture prepared as described above. The specifically bound cpm obtained were fit to a one-site binding model using GraphPad Prism ver. 2.0. Estimated IC50 values were converted to Ki values using the Cheng-Prusoff equation (Cheng, Y. C. et al., Biochem. Pharmacol., 22, 3099-108, 1973). The Ki values obtained from the assay are shown in Table 1.